Clotting of blood is a complicated process involving a large number of blood components including fibrinogen and prothrombin which is converted to thrombin. It has long been recognized that many aspects of unexplained bleeding or abnormal clotting can be explained in terms of improper levels of these materials in the blood. For instance, states of hypo-fibrinogenemia or hyper-fibrinogenemia may result from hepatic disease, from disseminated intravascular coagulation, from fibrinolytic syndrome, neoplastic disease, and post-operatively due to trauma. By monitoring the fibrinogen, thrombin and prothrombin levels within the blood, a physician may acquire meaningful data concerning the patient's blood clotting abilities. For example, the Activated Partial Thromboplastin Time (APTT) Test measures coagulation factors of the intrinsic pathway. These factors include Factors XII, XI, IX, VIII, X, V, II and I which may be abnormal based on heredity or heparin therapy. Thus, the APTT test is useful as a presurgical screen and for monitoring heparin therapy. Similarly, fibrinogen testing (by the Thrombin Time (TT) test or quantitative fibrinogen test) provides useful diagnostic data when unexplained bleeding or abnormal clotting occurs.
Within the past decade, evidence has accumulated linking soluble clotting factors with ischemic heart disease. Studies have shown that elevated blood plasma concentrations of Factors VIIa, VIIIc, and fibrinogen appear to be associated with an increased risk of heart attack and cardiovascular death. Patients who are in such a hypercoagulable state require careful monitoring. In the past, decisions to change medication, adjust dosage, or proceed with surgery were made based upon clotting time.
The process of blood coagulation occurs as a series of complex steps which terminate in the formation of a fibrin clot. Clot formation may occur by activation of the intrinsic pathway. In this system, coagulation factors circulate in the form of inactive precursors which are converted into an active form, which in turn activates the next clotting factor in sequence, i.e., proenzyme Factor XII is converted to its enzyme XIIa which in turn converts the zymogen Factor XI to the enzyme Factor XIa, which then activates Factor IX, in the presence of calcium. The enzyme Factor IXa in the presence of Factor VIII and phospholipid activates Factor X. This reaction is greatly increased by the prior exposure of Factor VIII to thrombin or Factor Xa.
In the extrinsic pathway, Factor X can be activated by either a complex of thromboplastin and Factor VII, or a complex of platelet phospholipid activated Factor IX and Factor VIII. Activated Factor X, in the presence of calcium, Factor V and platelet phospholipid activates Factor II (prothrombin) which is cleaved to form thrombin which converts Factor I (fibrinogen) to fibrin in blood plasma.
The process of blood coagulation is modified by a number of positive and negative feed back loops and by interaction between these pathways. For example, thrombin and Factor Xa, formed either by activation of the intrinsic or extrinsic pathway, feed back to activate Factor VIII and Factor V. Factor Xa feeds back to initially increase and then to inhibit its own activation by Factor VIIa. The intrinsic and extrinsic pathways are also linked. For example, Factor VII is activated by Factor IXa, XIIa and XIa and Factor VIIa can activate Factor IX.
The basis of in vitro coagulation testing has been the determination of the increase in turbidity or viscosity of a sample, caused by the conversion of fibrinogen to fibrin during clot formation. The screening tests for coagulation disorders include the prothrombin time (PT) and the activated partial thromboplastin time (APTT). Essentially, the screening tests for coagulation disorders are designed to detect a significant abnormality in one or more of the clotting factors and to localize this abnormality to various steps in the coagulation pathway. For example, APTT measures coagulation factors of the intrinsic pathway, including Factors XII, XI, IX, VIII, X, V, II and I which may be abnormal based on heredity or heparin therapy. APTT is therefore useful as a presurgical screen and for monitoring heparin therapy.
The APTT is performed by adding an activator such as kaolin, ellagic acid, or silica, for example, with phospholipid to plasma. This activates Factors XII and XI. Phospholipid substitutes for platelet in the activation of Factor VIII by Factors IX, VIII and V. Blood coagulation is initiated in this clotting test by adding calcium. Factor VII is the only factor not affected by the partial thromboplastin time and the APTT is, therefore, normal in patients with a Factor VII deficiency.
The prothrombin time (PT) test is performed by adding tissue thromboplastin with calcium to plasma. This initiates clotting by activating Factor VII which in turn activates Factor X which in the presence of Factor V, converts prothrombin to thrombin and the thrombin which is so produced converts fibrinogen to fibrin. PT therefore bypasses the intrinsic clotting pathway and is normal in patients with deficiencies of Factors XII, XI, IX and VIII. PT is abnormal in patients with deficiencies of Factors VII, X, V, prothrombin or fibrinogen.
As a result, substantial efforts have been made to measure these clotting components. Most methodologies rely upon immunologic and clotting techniques although clearly the latter is preferred. The immunologic techniques, although generally capable of precisely defining the levels of the various components within the blood stream, are incapable of distinguishing between active and inactive forms. Accordingly, the immunologic methods are felt to be less accurate with respect to the patient's actual clotting ability. Consequently, the results obtained by clotting techniques are preferred as being more clinically significant.
The human eye was the first clot detection system used for coagulation testing, e.g., a normal sample produces a strong gel clot; samples producing thin, watery, webby-type clots are indicative of some coagulation abnormality. Automated coagulation instrumentation, both mechanical and optical density-based, provide data about the end point of the clotting times in the various coagulation tests, e.g., PT and APTT. Typically, most instruments detect the formation of a clot by monitoring either optical turbidity or electrical conductivity. The latter represents the traditional approach employed by the so-called fibrometer-type of instrument. Effectively, this instrument measures increasing conductivity which may be correlated to the formation of clots. Similarly, turbidity may be optically sensed by the decrease in light transmission due to the formation of a clot. Certainly with the normal PT or APTT tests, these methods have found widespread acceptance despite the fact that each test has associated therewith a level of indefiniteness regarding the point at which the clot is determined to have occurred.
A more advanced instrument, such as the KoaguLab.RTM. (Ortho Diagnostic Systems Inc., Raritan, N.J.) generates a printed graph of the clotting reaction. Clinicians can tell by the shape of the curve generated whether or not the clotting times is reliable, thus providing a stronger information base for their therapeutic decisions. A graph which plots turbidity against reaction time is referred to as "clot signature". As used in the KoaguLab.RTM. system, the total turbidity change attached at the maximum clotting endpoint is "delta", the reaction rate of the observed clot formation is Velocity, and the derivative of Velocity or the maximum acceleration of observed clot formation is Acceleration. KoaguLab.RTM. may be used to perform PT and APTT assays. These are performed be adding brain thromboplastin or activated partial thromboplastin and calcium chloride respectively, to a plasma sample and determining the time at which the clot forms. Reagents useful for these purposes include, for instance, Ortho Quantitative Fibrinogen Assay (Q.F.A.), Ortho Q.F.A. Thrombin (Human), Ortho Q.F.A. Buffer, Ortho Activated PTT Reagent, Ortho Activated Thrombofax.TM. Reagent, Ortho Brain Thromboplastin, Fibrindex.TM.Thrombin, Ortho Plasma Coagulation Controls (obtainable from Ortho Diagnostic Systems Inc., Raritan, N.J.). These materials are accompanied by procedural instructions regarding their use, the relevant portions of which are incorporated herein by reference. Either of the two generalized embodiments of the methods of the present invention may be utilized for calculating PT or APTT coagulation times.
The clot signature essentially adds a qualitative fibrinogen measurement to the standard PT and APTT tests, which may prove useful in detecting certain disease states, including hypercoagulability.